BDNF induces structural and functional changes in central neurons to modulate synaptic efficacy; our goal is to identify molecular mechanisms that regulate BDNF targeting and release at synapses to modulate neurotransmission. BDNF is synthesized as a precursor, proBDNF, sorted to a regulated secretory pathway, and released in an activity-dependent manner. At the synapse, proBDNF can bind selectively to p75 to induce LTD, and potentially reduce spine density and dendritic complexity. If proBDNF is converted to mature BDNF in the secretory vesicle or synaptic cleft, TrkB is selectively activated to enhance synaptic transmission and promote axonal branching and dendritic growth. Thus, mechanisms that regulate conversion of proBDNF to mature BDNF, and regulate trafficking to dendrites or axons critically modulate structural and functional neuronal plasticity. We have generated knock-in mice expressing HA-tagged BDNF to markedly enhance detection of endogenous BDNF. We have also identified intracellular chaperones, including sortilin, and other sortilin family members that bind proBDNF. With these tools, three interrelated aims are proposed: (1) Using neurons from the BDNF-HA mouse, identify if conversion of proBDNF to mature BDNF occurs during sorting to secretory vesicles, or following vesicle fusion and release. We postulate that the location of BDNF conversion may differ among neuronal subtypes. (2) We will identify the sortilin family members that chaperone proBDNF to the constitutive or regulated secretory pathways, and to dendrites or axons. We posit that different sortilin members direct intracellular trafficking to different subcellular compartments, delivery to the synapse, and regulate cleavage to mature BNDF. Using BDNF-HA mouse, and acute silencing of different chaperones, we will assess the developmentally regulated changes in the ratio of proBDNF/mature BDNF release, and in retrograde and anterograde traffiking of BDNF isoforms. (3) We will generate knock-in mice to conditionally delete relevant sortilin family members. These animals will permit us to dissect the roles of select BDNF chaperones in regulating BDNF levels, targeting to axons or dendrites, and effects on neuronal morphology and connectively in the intact, postnatal brain. RELEVANCE (See instructions): This project identifies mechanisms that regulate BDNF release, and modulates morphology and connectivity of hippocampal and cortical neurons; disregulation of these processes contributes to neurodevelopmental disease. A human SNP that reduces BDNF release results in anxiety and depressive symptoms in mice, and correlates with these human diseases. Abnormal frontolimbic connectivity underiies conditions of anxiety anri autism Ths mfjchanlRms irifintifiert hfirR will vieiri npw tarnRt.; fnr fixaminatinn in thRSfi riiseases